Chronic lymphocytic leukemia cell line

ABSTRACT

The preparation and characterization of antibodies that bind to antigens on CLL or other cancer cells, especially to antigens upregulated in the cancer cells, and the identification and characterization of antigens present on or upregulated by cancer cells are useful in studying and treating cancer.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/286,759, filed Sep. 30, 2008, which is a continuation of U.S.application Ser. No. 10/379,151, filed Mar. 4, 2003, which is acontinuation in part of PCT/US01/47931 filed on Dec. 10, 2001 which isan international application that claims priority to U.S. ProvisionalApplication No. 60/254,113 filed Dec. 8, 2000. The entire disclosures ofthe aforementioned applications are incorporated herein by reference.

TECHNICAL FIELD

Cell lines derived from chronic lymphocytic leukemia (CLL) cells and theuses thereof in the study and treatment of CLL disease are disclosed. Inparticular, this disclosure relates to a CLL cell line designated“CLL-AAT”, deposited on Dec. 11, 2001 with the American Type CultureCollection (Manassas, Va., USA) in accordance with the terms of theBudapest Treaty under ATCC accession no. PTA-3920.

BACKGROUND

Chronic Lymphocytic Leukemia (CLL) is a disease of the white blood cellsand is the most common form of leukemia in the Western Hemisphere. CLLrepresents a diverse group of diseases relating to the growth ofmalignant lymphocytes that grow slowly but have an extended life span.CLL is classified in various categories that include, for example,B-cell chronic lymphocytic leukemia (B-CLL) of classical and mixedtypes, B-cell and T-cell prolymphocytic leukemia, hairy cell leukemia,and large granular lymphocytic leukemia.

Of all the different types of CLL, B-CLL accounts for approximately 30percent of all leukemias. Although it occurs more frequently inindividuals over 50 years of age it is increasingly seen in youngerpeople. B-CLL is characterized by accumulation of B-lymphocytes that aremorphologically normal but biologically immature, leading to a loss offunction. Lymphocytes normally function to fight infection. In B-CLL,however, lymphocytes accumulate in the blood and bone marrow and causeswelling of the lymph nodes. The production of normal bone marrow andblood cells is reduced and patients often experience severe anemia aswell as low platelet counts. This can pose the risk of life-threateningbleeding and the development of serious infections because of reducednumbers of white blood cells.

To further understand diseases such as leukemia it is important to havesuitable cell lines that can be used as tools for research on theiretiology, pathogenesis and biology. Examples of malignant humanB-lymphoid cell lines include pre-B acute lymphoblasticleukemia (Reh),diffuse large cell lymphoma (WSU-DLCL2), and Waldenstrom'smacroglobulinemia (WSU-WM). Unfortunately, many of the existing celllines do not represent the clinically most common types of leukemia andlymphoma.

The use of Epstein Barr Virus (EBV) infection in vitro has resulted insome CLL derived cell lines, in particular B-CLL cells lines, that arerepresentative of the malignant cells. The phenotype of these cell linesis different than that of the in vivo tumors and instead the features ofB-CLL lines tend to be similar to those of Lymphoblastoid cell lines.Attempts to immortalize B-CLL cells with the aid of EBV infection havehad little success. The reasons for this are unclear but it is knownthat it is not due a lack of EBV receptor expression, binding or uptake.Wells et al. found that B-CLL cells were arrested in the G1/S phase ofthe cell cycle and that transformation associated EBV DNA was notexpressed. This suggests that the interaction of EBV with B-CLL cells isdifferent from that with normal B cells. EBV-transformed CLL cell linesmoreover appear to differentiate, possessing a morphology more similarto lymphoblastoid cell lines (LCL) immortalized by EBV.

An EBV-negative CLL cell line, WSU-CLL, has been established previously(Mohammad et al., (1996) Leukemia 10(1):130-7). However, no other suchcell lines are known.

There remains a need in the art, therefore, for a CLL cell line whichhas not been established by transformation with EBV, and which expressessurface markers characteristic of primary CLL cells.

SUMMARY

In one embodiment an CLL cell line of malignant origin is provided thatis not established by immortalisation with EBV. The cell line, which wasderived from primary CLL cells, and is deposited under ATCC accessionno. PTA-3920. In a preferred embodiment, the cell line is CLL-AAT.CLL-AAT is B-CLL cell line, derived from a B-CLL primary cell.

In a further aspect, the CLL-AAT cell line is used to generatemonoclonal antibodies useful in the diagnosis and/or treatment of CLL.Antibodies may be generated by using the cells as disclosed herein asimmunogens, thus raising an immune response in animals from whichmonoclonal antibodies may be isolated. The sequence of such antibodiesmay be determined and the antibodies or variants thereof produced byrecombinant techniques. In this aspect, “variants” includes chimeric,CDR-grafted, humanized and fully human antibodies based on the sequenceof the monoclonal antibodies.

Moreover, antibodies derived from recombinant libraries (“phageantibodies”) may be selected using the cells described herein, orpolypeptides derived therefrom, as bait to isolate the antibodies on thebasis of target specificity.

In a still further aspect, antibodies may be generated by panningantibody libraries using primary CLL cells, or antigens derivedtherefrom, and further screened and/or characterized using a CLL cellline, such as, for example, the CLL cell line described herein.Accordingly, a method for characterizing an antibody specific for CLL isprovided, which includes assessing the binding of the antibody to a CLLcell line.

In a further aspect, there is provided a method for identifying proteinsuniquely expressed in CLL cells employing the CLL-AAT cell line, bymethods well known to those, skilled with art, such as byimmunoprecipitation followed by mass spectroscopy analyses. Suchproteins may be uniquely expressed in the CLL-AAT cell line, or inprimary cells derived from CLL patients.

Small molecule libraries (many available commercially) may be screenedusing the CLL-AAT cell line in a cell-based assay to identify agentscapable of modulating the growth characteristics of the cells. Forexample, the agents may be identified which modulate apoptosis in theCLL-AAT cell line, or which inhibit growth and/or proliferation thereof.Such agents are candidates for the development of therapeutic compounds.

Nucleic acids isolated from CLL-AAT cell lines may be used insubtractive hybridization experiments to identify CLL-specific genes orin micro array analyses (e.g., gene chip experiments). Genes whosetranscription is modulated in CLL cells may be identified. Polypeptideor nucleic acid gene products identified in this manner are useful asleads for the development of antibody or small molecule therapies forCLL.

In a preferred aspect, the CLL-AAT cell line may be used to identifyinternalizing antibodies, which bind to cell surface components whichare internalized by the cell. Such antibodies are candidates fortherapeutic use. In particular, single-chain antibodies, which remainstable in the cytoplasm and which retain intracellular binding activity,may be screened in this manner.

In yet another aspect, a therapeutic treatment is described in which apatient is screened for the presence of a polypeptide that isupregulated by a malignant cancer cell and an antibody that interfereswith the metabolic pathway of the upregulated polypeptide isadministered to the patient.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. schematically illustrates typical steps involved in cell surfacepanning of antibody libraries by magnetically-activated cell sorting(MACS).

FIG. 2. is a graph showing the results of whole cell ELISA demonstratingbinding of selected scFv clones to primary B-CLL cells and absence ofbinding to normal human PBMC. The designation 2°+3° in this and otherfigures refers to negative control wells stained with Mouse Anti-HA anddetecting antimouse antibodies alone. The designation RSC-S Library inthis and other figures refers to soluble antibodies prepared fromoriginal rabbit scFv unpanned library. The designation R3/RSC-S Pool inthis and other figures refers to soluble antibodies prepared from entirepool of scFv antibodies from round 3 of panning Anti-CD5 antibody wasused as a positive control to verify that equal numbers of B-CLL andPBMC cells were plated in each well.

FIGS. 3 a and 3 b show the results of whole cell ELISA comparing bindingof selected scFv antibodies to primary B-CLL cells and normal primaryhuman B cells. Anti-CD19 antibody was used as a positive control toverify that equal numbers of B-CLL and normal B cells were plated ineach well. Other controls were as described in the legend to FIG. 2.

FIGS. 4 a and 4 b show the results of whole cell ELISA used to determineif scFv clones bind to patient-specific (i.e. idiotype) or bloodtype-specific (i.e. HLA) antigens. Each clone was tested for binding toPBMC isolated from 3 different B-CLL patients. Clones that bound to ≦1patient sample were considered to be patient or blood type-specific.

FIGS. 5 a and 5 b show the results of whole cell ELISA comparing bindingof scFv clones to primary B-CLL cells and three human leukemic celllines. Ramos is a mature B cell line derived from a Burkitt's lymphoma.RL is a mature B cell line derived from a non-Hodgkin's lymphoma. TF-Iis an erythroblastoid cell line derived from a erythroleukemia.

FIGS. 6 a, 6 b and 6 c show the results of whole cell ELISA comparingbinding of scFv clones to primary B-CLL cells and CLL-AAT, a cell linederived from a B-CLL patient. TF-I cells were included as a negativecontrol.

FIG. 7 shows the binding specificity of scFv antibodies in accordancewith this disclosure as analyzed by 3-color flow cytometry. In normalperipheral blood mononuclear cells, the antigen recognized by scFv-9 ismoderately expressed on B lymphocytes and weakly expressed on asubpopulation of T lymphocytes. PBMC from a normal donor were analyzedby 3-color flow cytometry using anti-CD5-FITC, anti-CD19-PerCP, andscFv-9/Anti-HA-biotin/streptavidin-PE.

FIGS. 8 a, 8 b and 8 c show the expression levels of antigens recognizedby scFv antibodies in accordance with this disclosure. The antigensrecognized by scFv-3 and scFv-9 are overexpressed on the primary CLLtumor from which the CLL-AAT cell line was derived. Primary PBMC fromthe CLL patient used to establish the CLL-AAT cell line or PBMC from anormal donor were stained with scFv antibody and analyzed by flowcytometry. ScFv-3 and scFv-9 stain the CLL cells more brightly than thenormal PBMC as measured by the mean fluorescent intensities.

FIGS. 9 a-9 c provide a summary of CDR sequences and bindingspecificities of selected scFv antibodies.

FIG. 10. is Table 2 which shows a summary of flow cytometry resultscomparing expression levels of scFv antigens on primary CLL cells vs.normal PBMC as described in FIGS. 8 a-8 c.

FIG. 11. is a Table showing a summary of flow cytometry resultscomparing expression levels of scFv-9 antigen with the percentage ofCD38+ cells in peripheral blood mononuclear cells isolated from ten CLLpatients.

FIG. 12. shows the identification of scFv antigens byimmunoprecipitation and mass spectrometry. CLL-AAT cells were labeledwith a solution of 0.5 mg/ml sulfo-NHS-LC-biotin (Pierce) in PBS, pH8.0for 30′. After extensive washing with PBS to remove unreacted biotin,the cells were disrupted by nitrogen cavitation and the microsomalfraction was isolated by differential centrifugation. The microsomalfraction was resuspended in NP40 Lysis Buffer and extensively preclearedwith normal rabbit serum and protein A sepharose. Antigens wereimmunoprecipitated with HA-tagged scFv antibodies coupled to Rat Anti-HAagarose beads (Roche). Following immunoprecipitation, antigens wereseparated by SDS-PAGE and detected by Western blot usingstreptavidin-alkaline phosphatase (AP) or by Coomassie G-250 stainingScFv-7, an antibody which doesn't bind to CLL-AAT cells, was used as anegative control. Antigen bands were excised from the Coomassie-stainedgel and identified by mass spectrometry (MS). MALDI-MS was performed atthe Proteomics Core Facility of The Scripps Research Institute (LaJolla, Calif.). μLC/MS/MS was performed at the Harvard MicrochemistryFacility (Cambridge, Mass.).

FIG. 13. shows that three scFv antibodies bind specifically to 293-EBNAcells transiently transfected with a human OX-2/CD200 cDNA clone. ACD200 cDNA was cloned from CLL cells by RT-PCR and inserted into themammalian expression vector pCEP4 (Invitrogen). PCEP4-CD200 plasmid orthe corresponding empty vector pCEP4 was transfected into 293-EBNA cellsusing Polyfect reagent (QIAGEN). Two days after transfection, the cellswere analyzed for binding to scFv antibodies by flow cytometry.

DETAILED DESCRIPTION Definitions

“CLL”, as used herein, refers to chronic lymphocytic leukemia involvingany lymphocyte, including but not limited to various developmentalstages of B cells and T cells, including but not limited to B cell CLL.B-CLL, as used herein, refers to leukemia with a mature B cell phenotypewhich is CD5⁺, CD23⁺, CD2O^(dim+), sIg^(dim+) and arrested in GO/G1 ofthe cell cycle.

“Malignant origin” refers to the derivation of the cell line frommalignant CLL primary cells, as opposed to non-proliferating cells whichare transformed, for example, with EBV. Cell lines according to thisdisclosure may be themselves malignant in phenotype, or not. A CLL cellhaving a “malignant” phenotype encompasses cell growth unattached fromsubstrate media characterized by repeated cycles of cell growth andexhibits resistance to apoptosis.

Preparation of Cell Lines

Cell lines may be produced according to established methodologies knownto those skilled in the art. In general, cell lines are produced byculturing primary cells derived from a patient until immortalized cellsare spontaneously generated in culture. These cells are then isolatedand further cultured, to produce clonal cell populations or cellsexhibiting resistance to apoptosis.

For example, CLL cells may be isolated from peripheral blood drawn froma patient suffering from CLL. The cells may be washed, and optionallyimmunotyped in order to determine the type(s) of cells present.Subsequently, the cells may be cultured in a medium, such as a mediumcontaining IL-4. Advantageously, all or part of the medium is replacedone or more times during the culture process. Cell lines may be isolatedthereby, and will be identified by increased growth in culture.

Preparation of Monoclonal Antibodies

Antibodies, as used herein, refers to complete antibodies or antibodyfragments capable of binding to a selected target. Included are Fv,ScFv, Fab′ and F(ab′)₂, monoclonal and polyclonal antibodies, engineeredantibodies (including chimeric, CDR-grafted and humanized, fully humanantibodies, and artificially selected antibodies), and synthetic or semisynthetic antibodies produced using phage display or alternativetechniques. Small fragments, such Fv and ScFv, possess advantageousproperties for diagnostic and therapeutic applications on account oftheir small size and consequent superior tissue distribution.

The antibodies are especially indicated for diagnostic and therapeuticapplications. Accordingly, they may be altered antibodies comprising aneffector protein such as a toxin or a label. Especially preferred arelabels which allow the imaging of the distribution of the antibody invivo. Such labels may be radioactive labels or radiopaque labels, suchas metal particles, which are readily visualisable within the body of apatient. Moreover, the labels may be fluorescent labels or other labelswhich are visualisable on tissue samples removed from patients.

Recombinant DNA technology may be used to improve the antibodiesproduced in accordance with this disclosure. Thus, chimeric antibodiesmay be constructed in order to decrease the immunogenicity thereof indiagnostic or therapeutic applications. Moreover, immunogenicity may beminimized by humanizing the antibodies by CDR grafting and, optionally,framework modification. See, U.S. Pat. No. 5,225,539, the contents ofwhich are incorporated herein by reference.

Antibodies may be obtained from animal serum, or, in the case ofmonoclonal antibodies or fragments thereof produced in cell culture.Recombinant DNA technology may be used to produce the antibodiesaccording to established procedure, in bacterial or preferably mammaliancell culture. The selected cell culture system preferably secretes theantibody product.

In another embodiment, a process for the production of an antibodydisclosed herein includes culturing a host, e.g. E. coli or a mammaliancell, which has been transformed with a hybrid vector. The vectorincludes one or more expression cassettes containing a promoter operablylinked to a first DNA sequence encoding a signal peptide linked in theproper reading frame to a second DNA sequence encoding the antibodyprotein. The antibody protein is then collected and isolated.Optionally, the expression cassette may include a promoter operablylinked to polycistronic, for example bicistronic, DNA sequences encodingantibody proteins each individually operably linked to a signal peptidein the proper reading frame.

Multiplication of hybridoma cells or mammalian host cells in vitro iscarried out in suitable culture media, which include the customarystandard culture media (such as, for example Dulbecco's Modified EagleMedium (DMEM) or RPMI 1640 medium), optionally replenished by amammalian serum (e.g. fetal calf serum), or trace elements and growthsustaining supplements (e.g. feeder cells such as normal mouseperitoneal exudate cells, spleen cells, bone marrow macrophages,2-aminoethanol, insulin, transferrin, low density lipoprotein, oleicacid, or the like). Multiplication of host cells which are bacterialcells or yeast cells is likewise carried out in suitable culture mediaknown in the art. For example, for bacteria suitable culture mediainclude medium LE, NZCYM, NZYM, NZM, Terrific Broth, SOB, SOC, 2×YT, orM9 Minimal Medium. For yeast, suitable culture media include medium YPD,YEPD, Minimal Medium, or Complete Minimal Dropout Medium.

In vitro production provides relatively pure antibody preparations andallows scale-up to give large amounts of the desired antibodies.Techniques for bacterial cell, yeast, plant, or mammalian cellcultivation are known in the art and include homogeneous suspensionculture (e.g. in an airlift reactor or in a continuous stirrer reactor),and immobilized or entrapped cell culture (e.g. in hollow fibres,microcapsules, on agarose microbeads or ceramic cartridges).

Large quantities of the desired antibodies can also be obtained bymultiplying mammalian cells in vivo. For this purpose, hybridoma cellsproducing the desired antibodies are injected into histocompatiblemammals to cause growth of antibody-producing tumors. Optionally, theanimals are primed with a hydrocarbon, especially mineral oils such aspristane (tetramethyl-pentadecane), prior to the injection. After one tothree weeks, the antibodies are isolated from the body fluids of thosemammals. For example, hybridoma cells obtained by fusion of suitablemyeloma cells with antibody-producing spleen cells from Balb/c mice, ortransfected cells derived from hybridoma cell line Sp2/0 that producethe desired antibodies are injected intraperitoneally into Balb/c miceoptionally pre-treated with pristine. After one to two weeks, asciticfluid is taken from the animals.

The foregoing, and other, techniques are discussed in, for example,Kohler and Milstein, (1975) Nature 256:495-497; U.S. Pat. No. 4,376,110;Harlow and Lane, Antibodies: a Laboratory Manual, (1988) Cold SpringHarbor, the disclosures of which are all incorporated herein byreference. Techniques for the preparation of recombinant antibodymolecules is described in the above references and also in, for exampleWO97/08320; U.S. Pat. No. 5,427,908; U.S. Pat. No. 5,508,717; Smith,1985, Science, Vol. 225, pp 1315-1317; Parmley and Smith 1988, Gene 73,pp 305-318; De La Cruz et al, 1988, Journal of Biological Chemistry, 263pp 4318-4322; U.S. Pat. No. 5,403,484; U.S. Pat. No. 5,223,409;WO88/06630; WO92/15679; U.S. Pat. No. 5,780,279; U.S. Pat. No.5,571,698; U.S. Pat. No. 6,040,136; Davis et al., Cancer MetastasisRev., 1999; 18(4):421-5; Taylor, et al., Nucleic Acids Research 20(1992): 6287-6295; Tomizuka et al., Proc. Nat. Academy of Sciences USA97(2) (2000): 722-727. The contents of all these references areincorporated herein by reference.

The cell culture supernatants are screened for the desired antibodies,preferentially by immunofluorescent staining of CLL cells, byimmunoblotting, by an enzyme immunoassay, e.g. a sandwich assay or adot-assay, or a radioimmunoassay.

For isolation of the antibodies, the immunoglobulins in the culturesupernatants or in the ascitic fluid may be concentrated, e.g. byprecipitation with ammonium sulfate, dialysis against hygroscopicmaterial such as polyethylene glycol, filtration through selectivemembranes, or the like. If necessary and/or desired, the antibodies arepurified by the customary chromatography methods, for example gelfiltration, ion-exchange chromatography, chromatography overDEAE-cellulose and/or (immuno-) affinity chromatography, e.g. affinitychromatography with a one or more surface polypeptides derived from aCLL cell line according to this disclosure, or with Protein-A or G.

Another embodiment provides a process for the preparation of a bacterialcell line secreting antibodies directed against the cell linecharacterized in that a suitable mammal, for example a rabbit, isimmunized with pooled CLL patient samples. A phage display libraryproduced from the immunized rabbit is constructed and panned for thedesired antibodies in accordance with methods well known in the art(such as, for example, the methods disclosed in the various referencesincorporated herein by reference).

Hybridoma cells secreting the monoclonal antibodies are alsocontemplated. The preferred hybridoma cells are genetically stable,secrete monoclonal antibodies described herein of the desiredspecificity and can be activated from deep-frozen cultures by thawingand recloning.

In another embodiment, a process is provided for the preparation of ahybridoma cell line secreting monoclonal antibodies directed to the CLLcell line is described herein. In that process, a suitable mammal, forexample a Balb/c mouse, is immunized with a one or more polypeptides orantigenic fragments thereof derived from a cell described in thisdisclosure, the cell line itself, or an antigenic carrier containing apurified polypeptide as described. Antibody-producing cells of theimmunized mammal are grown briefly in culture or fused with cells of asuitable myeloma cell line. The hybrid cells obtained in the fusion arecloned, and cell clones secreting the desired antibodies are selected.For example, spleen cells of Balb/c mice immunized with the present cellline are fused with cells of the myeloma cell line PAI or the myelomacell line Sp2/0-Ag 14, the obtained hybrid cells are screened forsecretion of the desired antibodies, and positive hybridoma cells arecloned.

Preferred is a process for the preparation of a hybridoma cell line,characterized in that Balb/c mice are immunized by injectingsubcutaneously and/or intraperitoneally between 10⁶ and 10⁷ cells of acell line in accordance with this disclosure several times, e.g. four tosix times, over several months, e.g. between two and four months. Spleencells from the immunized mice are taken two to four days after the lastinjection and fused with cells of the myeloma cell line PAI in thepresence of a fusion promoter, preferably polyethylene glycol.Preferably, the myeloma cells are fused with a three- to twenty-foldexcess of spleen cells from the immunized mice in a solution containingabout 30% to about 50% polyethylene glycol of a molecular weight around4000. After the fusion, the cells are expanded in suitable culture mediaas described hereinbefore, supplemented with a selection medium, forexample HAT medium, at regular intervals in order to prevent normalmyeloma cells from overgrowing the desired hybridoma cells.

In a further embodiment, recombinant DNA comprising an insert coding fora heavy chain variable domain and/or for a light chain variable domainof antibodies directed to the cell line described hereinbefore areproduced. The term DNA includes coding single stranded DNAs, doublestranded DNAs consisting of said coding DNAs and of complementary DNAsthereto, or these complementary (single stranded) DNAs themselves.

Furthermore, DNA encoding a heavy chain variable domain and/or a lightchain variable domain of antibodies directed to the cell line disclosedherein can be enzymatically or chemically synthesized DNA having theauthentic DNA sequence coding for a heavy chain variable domain and/orfor the light chain variable domain, or a mutant thereof. A mutant ofthe authentic DNA is a DNA encoding a heavy chain variable domain and/ora light chain variable domain of the above-mentioned antibodies in whichone or more amino acids are deleted or exchanged with one or more otheramino acids. Preferably said modification(s) are outside the CDRs of theheavy chain variable domain and/or of the light chain variable domain ofthe antibody in humanization and expression optimization applications.The term mutant DNA also embraces silent mutants wherein one or morenucleotides are replaced by other nucleotides with the new codons codingfor the same amino acid(s). The term mutant sequence also includes adegenerated sequence. Degenerated sequences are degenerated within themeaning of the genetic code in that an unlimited number of nucleotidesare replaced by other nucleotides without resulting in a change of theamino acid sequence originally encoded. Such degenerated sequences maybe useful due to their different restriction sites and/or frequency ofparticular codons which are preferred by the specific host, particularlyE. coli, to obtain an optimal expression of the heavy chain murinevariable domain and/or a light chain murine variable domain.

The term mutant is intended to include a DNA mutant obtained by in vitromutagenesis of the authentic DNA according to methods known in the art.

For the assembly of complete tetrameric immunoglobulin molecules and theexpression of chimeric antibodies, the recombinant DNA inserts codingfor heavy and light chain variable domains are fused with thecorresponding DNAs coding for heavy and light chain constant domains,then transferred into appropriate host cells, for example afterincorporation into hybrid vectors.

Recombinant DNAs including an insert coding for a heavy chain murinevariable domain of an antibody directed to the cell line disclosedherein fused to a human constant domain g, for example γ1, γ2, γ3 or γ4,preferably γ1 or γ4 are also provided. Recombinant DNAs including aninsert coding for a light chain murine variable domain of an antibodydirected to the cell line disclosed herein fused to a human constantdomain κ or λ, preferably κ are also provided

Another embodiment pertains to recombinant DNAs coding for a recombinantpolypeptide wherein the heavy chain variable domain and the light chainvariable domain are linked by way of a spacer group, optionallycomprising a signal sequence facilitating the processing of the antibodyin the host cell and/or a DNA coding for a peptide facilitating thepurification of the antibody and/or a cleavage site and/or a peptidespacer and/or an effector molecule.

The DNA coding for an effector molecule is intended to be a DNA codingfor the effector molecules useful in diagnostic or therapeuticapplications. Thus, effector molecules which are toxins or enzymes,especially enzymes capable of catalyzing the activation of prodrugs, areparticularly indicated. The DNA encoding such an effector molecule hasthe sequence of a naturally occurring enzyme or toxin encoding DNA, or amutant thereof, and can be prepared by methods well known in the art.

Antibodies and antibody fragments disclosed herein are useful indiagnosis and therapy. Accordingly, a composition for therapy ordiagnosis comprising an antibody disclosed herein is provided.

In the case of a diagnostic composition, the antibody is preferablyprovided together with means for detecting the antibody, which may beenzymatic, fluorescent, radioisotopic or other means. The antibody andthe detection means may be provided for simultaneous, separate orsequential use, in a diagnostic kit intended for diagnosis.

Uses of the CLL Cell Line

There are many advantages to the development of a CLL cell line, as itprovides an important tool for the development of diagnostics andtreatments for CLL.

A cell line according to this disclosure may be used for in vitrostudies on the etiology, pathogenesis and biology of CLL. This assistsin the identification of suitable agents that are useful in the therapyof CLL disease.

The cell line may also be used to produce monoclonal antibodies for invitro and in vivo diagnosis of CLL, as referred to above, and for thescreening and/or characterization of antibodies produced by othermethods, such as by panning antibody libraries with primary cells and/orantigens derived from CLL patients.

The cell line may be used as such, or antigens may be derived therefrom.Advantageously, such antigens are cell-surface antigens specific forCLL. They may be isolated directly from cell lines according to thisdisclosure. Alternatively, a cDNA expression library made from a cellline described herein may be used to express CLL-specific antigens,useful for the selection and characterization of anti-CLL antibodies andthe identification of novel CLL-specific antigens.

Treatment of CLL using monoclonal antibody therapy has been proposed inthe art. Recently, Hainsworth (Oncologist 5 (5) (2000) 376-384) hasdescribed the current therapies derived from monoclonal antibodies.Lymphocytic leukemia in particular is considered to be a good candidatefor this therapeutic approach due to the presence of multiplelymphocyte-specific antigens on lymphocyte tumors.

Existing antibody therapies (such as Rituximab™, directed against theCD20-antigen, which is expressed on the surface of B-lymphocytes) havebeen used successfully against certain lymphocytic disease. However, alower density CD20 antigen is expressed on the surface of B-lymphocytesin CLL (Almasri et al., Am. J. Hematol., 40 (4) (1992) 259-263).

The CLL cell line described herein thus permits the development of novelanti-CLL antibodies having specificity for one or more antigenicdeterminants of the present CLL cell line, and their use in the therapyand diagnosis of CLL.

In a particularly useful embodiment, the antibody binds to or otherwiseinterferes with the metabolic pathway of a polypeptide that isupregulated by a malignant cancer cell. For instance, the antibody canbind to the upregulated polypeptide and in this manner prevent orinhibit the polypeptide from interacting with other molecules orreceptors. Alternatively, the antibody may bind to a receptor with whichthe upregulated polypeptide normally interacts, thereby preventing orinhibiting the polypeptide from binding to the receptor. As yet anotheralternative, the antibody can bind to an antigen that modulatesexpression of the polypeptide, thereby preventing or inhibiting normalor increased expression of the polypeptide. For example, the peptideOX-2/CD200 is upregulated in a portion of CLL patients. Because thepresence of OX-2/CD200 has been associated with reduced immune response,it would be desirable to interfere with the metabolic pathway ofOX-2/CD200 so that the patient's immune system can defend against thecancer more effectively.

Thus, in another embodiment, a method for treating a cancer patient inaccordance with this disclosure includes the steps of screening a cancerpatient for the presence of a polypeptide that is upregulated by amalignant cancer cell and administering an antibody that interferes withthe metabolic pathway of the upregulated polypeptide. In a particularlyuseful embodiment, a CLL patient is screened for overexpression ofOX-2/CD200 and an antibody that interferes with the metabolic pathway ofOX-2/CD200 is administered to the patient. As described in detail below,one such antibody is scFv9 (see FIG. 9B) which binds to OX-2/CD200.

In order that those skilled in the art may be better able to practicethe compositions and methods described herein, the following examplesare given for illustration purposes.

Example 1 Isolation of Cell Line CLL-AAT Establishment of the Cell Line

Peripheral blood from a patient diagnosed with CLL was obtained. The WBCcount was 1.6×10⁸/ml. Mononuclear cells were isolated by Histopaque-1077density gradient centrifugation (Sigma Diagnostics, St. Louis, Mo.).Cells were washed twice with Iscove's Modified Dulbecco's Medium (IMDM)supplemented with 10% heat-inactivated fetal bovine serum (FBS), andresuspended in 5 ml of ice-cold IMDM/10% FBS. Viable cells were countedby staining with trypan blue. Cells were mixed with an equal volume of85% FBS/15% DMSO and frozen in 1 ml aliquots for storage in liquidnitrogen.

Immunophenotyping showed that >90% of the CD45+ lymphocyte populationexpressed IgD, kappa light chain, CD5, CD19, and CD23. This populationalso expressed low levels of IgM and CD20. Approximately 50% of thecells expressed high levels of CD38. The cells were negative for lambdalight chain, CD10 and CD138

An aliquot of the cells was thawed, washed, and resuspended at a densityof 10⁷/mL in IMDM supplemented with 20% heat-inactivated FBS, 2 mML-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, 50 μM2-mercaptoethanol, and 5 ng/ml recombinant human IL-4 (R & D Systems,Minneapolis, Minn.). The cells were cultured at 37° C. in a humidified5% CO2 atmosphere. The medium was partially replaced every 4 days untilsteady growth was observed. After 5 weeks, the number of cells in theculture began to double approximately every 4 days. This cell line wasdesignated CLL-AAT.

Characterization of the Cell Line

Immunophenotyping of the cell line by flow cytometry showed highexpression of IgM, kappa light chain, CD23, CD38, and CD138, moderateexpression of CD19 and CD20, and weak expression of IgD and CD5. Thecell line was negative for lambda light chain, CD4, CD8, and CD10.

Immunophenotyping of the cell line was also done by whole cell ELISAusing a panel of rabbit scFv antibodies that had been selected forspecific binding to primary B-CLL cells. All of these CLL-specific scFyantibodies also recognized the CLL-AAT cell line. In contrast, themajority of the scFvs did not bind to two cell lines derived from B celllymphomas: Ramos, a Burkitt's lymphoma cell line, and RL, anon-Hodgkin's lymphoma cell line.

Example 2 Selection of scFv Antibodies for B-CLL-Specific Cell SurfaceAntigens Using Antibody Phage Display and Cell Surface Panning

Immunizations and scFv Antibody Library Construction

Peripheral blood mononuclear cells (PBMC) were isolated from blood drawnfrom CLL patients at the Scripps Clinic (La Jolla, Calif.). Two rabbitswere immunized with 2×10⁷ PBMC pooled from 10 different donors with CLL.Three immunizations, two sub-cutaneous injections followed by oneintravenous injection, were done at three week intervals. Serum titerswere checked by measuring binding of serum IgG to primary CLL cellsusing flow cytometry. Five days after the final immunization, spleen,bone marrow, and PBMC were harvested from the animals. Total RNA wasisolated from these tissues using Tri-Reagent (Molecular ResearchCenter, Inc). Single-chain Fv (scFv) antibody phage display librarieswere constructed as previously described (Barbas et al., (2001) PhageDisplay: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.). For cell surface panning, phagemid particles fromthe reamplified library were precipitated with polyethylene glycol(PEG), resuspended in phosphate-buffered saline (PBS) containing 1%bovine serum albumin (BSA), and dialysed overnight against PBS.

Antibody Selection by Cell Surface Panning

The libraries were enriched for CLL cell surface-specific antibodies bypositive-negative selection with a magnetically-activated cell sorter(MACS) as described by Siegel et al. (1997, J. Immunol. Methods206:73-85). Briefly, phagemid particles from the scFv antibody librarywere preincubated in MPBS (2% nonfat dry milk, 0.02% sodium azide inPBS, pH 7.4) for 1 hour at 25° C. to block nonspecific binding sites.Approximately 10⁷ primary CLL cells were labeled with mouse anti-CD5 IgGand mouse anti-CD19 IgG conjugated to paramagnetic microbeads (MiltenyiBiotec, Sunnyvale, Calif.). Unbound microbeads were removed by washing.The labeled CLL cells (“target cells”) were mixed with an excess of“antigen-negative absorber cells”, pelleted, and resuspended in 50 μl(10¹⁰-10¹¹ cfu) of phage particles. The absorber cells serve to soak upphage that stick non-specifically to cell surfaces as well as phagespecific for “common” antigens present on both the target and absorbercells. The absorber cells used were either TF-1 cells (a humanerythroleukemia cell line) or normal human B cells isolated fromperipheral blood by immunomagnetic negative selection (StemSep system,StemCell Technologies, Vancouver, Canada). The ratio of absorber cellsto target cells was approximately 10 fold by volume. After a 30 minuteincubation at 25° C., the cell/phage mixture was transferred to aMiniMACS MS+ separation column. The column was washed twice with 0.5 mlof MPBS, and once with 0.5 ml of PBS to remove the unbound phage andabsorber cells. The target cells were eluted from the column in 1 ml ofPBS and pelleted in a microcentrifuge at maximum speed for 15 seconds.The captured phage particles were eluted by resuspending the targetcells in 200 μl of acid elution buffer (0.1 N HCl, pH adjusted to 2.2with glycine, plus 1 μg/ml BSA). After a 10 minute incubation at 25° C.,the buffer was neutralized with 12 μL of 2M Tris base, pH10.5, and theeluted phage were amplified in E. coli for the next round of panning Foreach round of panning, the input and output phage titers weredetermined. The input titer is the number of reamplified phage particlesadded to the target cell/absorber cell mixture and the output titer isthe number of captured phage eluted from the target cells. An enrichmentfactor (E) is calculated using the formula E=(R_(n) output/R_(n)input)/(R_(l) output/R_(l) input). In most cases, an enrichment factorof 10²-10³ fold should be attained by the third or fourth round.

Analysis of Enriched Antibody Pools Following Panning

After 3-5 rounds of panning, the pools of captured phage were assayedfor binding to CLL cells by flow cytometry and/or whole cell ELISA:

-   1. To produce an entire pool in the form of HA-tagged soluble    antibodies, 2 ml of a non-suppressor strain of E. coli (e.g.    TOP1OF′) was infected with 1 μl (10⁹-10¹⁰ cfu) of phagemid    particles. The original, unpanned library was used as a negative    control. Carbenicillin was added to a final concentration of 10 μM    and the culture was incubated at 37° C. with shaking at 250 rpm for    1 hour. Eight ml of SB medium containing 50 μg/ml carbenicillin was    added and the culture was grown to an OD 600 of ˜0.8. IPTG was added    to a final concentration of 1 mM to induce scFv expression from the    Lac promoter and shaking at 37° C. was continued for 4 hours. The    culture was centrifuged at 3000×g for 15′. The supernatant    containing the soluble antibodies was filtered and stored in 1 ml    aliquots at −20° C.-   2. Binding of the scFv antibody pools to target cells vs. absorber    cells was determined by flow cytometry using high-affinity Rat    Anti-HA (clone 3F10, Roche Molecular Biochemicals) as secondary    antibody and PE-conjugated Donkey Anti-Rat as tertiary antibody.-   3. Binding of the antibody pools to target cells vs. absorber cells    was also determined by whole-cell ELISA as described below.    Screening Individual scFv Clones Following Panning

To screen individual scFv clones following panning, TOP1OF′ cells wereinfected with phage pools as described above, spread onto LB platescontaining carbenicillin and tetracycline, and incubated overnight at37° C. Individual colonies were inoculated into deep 96-well platescontaining 0.6-1.0 ml of SB-carbenicillin medium per well. The cultureswere grown for 6-8 hours in a HiGro shaking incubator (GeneMachines, SanCarlos, Calif.) at 520 rpm and 37° C. At this point, a 90 μl aliquotfrom each well was transferred to a deep 96-well plate containing 10 μLof DMSO. This replica plate was stored at −80° C. IPTG was added to theoriginal plate to a final concentration of 1 mM and shaking wascontinued for 3 hours. The plates were centrifuged at 3000×g for 15minutes. The supernatants containing soluble scFv antibodies weretransferred to another deep 96-well plate and stored at −20° C.

A sensitive whole-cell ELISA method for screening HA-tagged scFvantibodies was developed:

-   1. An ELISA plate is coated with concanavalin A (10 mg/ml in 0.1 M    NaHCO₃, pH8.6, 0.1 mM CaCl₂).-   2. After washing the plate with PBS, 0.5-1×10⁵ target cells or    absorber cells in 50 μl of PBS are added to each well, and the plate    is centrifuged at 250×g for 10 minutes.-   3. 50 μl of 0.02% glutaraldehyde in PBS are added and the cells are    fixed overnight at 4° C.-   4. After washing with PBS, non-specific binding sites are blocked    with PBS containing 4% non-fat dry milk for 3 hours at room    temperature.-   5. The cells are incubated with 50 μl of soluble, HA-tagged scFv or    Fab antibody (TOP10F′ supernatant) for 2 hours at room temperature,    then washed six times with PBS.-   6. Bound antibodies are detected using a Mouse Anti-HA secondary    antibody (clone 12CA5) and an alkaline phosphatase (AP)-conjugated    Anti-Mouse IgG tertiary antibody. An about 10-fold amplification of    the signal is obtained by using AMDEX AP-conjugated Sheep Anti-Mouse    IgG as the tertiary antibody (Amersham Pharmacia Biotech). The AMDEX    antibody is conjugated to multiple AP molecules via a dextran    backbone. Color is developed with the alkaline phosphatase substrate    PNPP and measured at 405 nm using a microplate reader.

Primary screening of the scFv clones was done by ELISA on primary CLLcells versus normal human PBMC. Clones which were positive on CLL cellsand negative on normal PBMC were rescreened by ELISA on normal human Bcells, human B cell lines, TF-1 cells, and the CLL-AAT cell line. Theclones were also rescreened by ELISA on CLL cells isolated from threedifferent patients to eliminate clones that recognized patient-specificor blood type-specific antigens. Results from representative ELISAs areshown in FIGS. 2-6 and summarized in FIG. 9.

The number of unique scFv antibody clones obtained was determined by DNAfingerprinting and sequencing. The scFv DNA inserts were amplified fromthe plasmids by PCR and digested with the restriction enzyme BstNI. Theresulting fragments were separated on a 4% agarose gel and stained withethidium bromide. Clones with different restriction fragment patternsmust have different amino acid sequences. Clones with identical patternsprobably have similar or identical sequences. Clones with unique BstNIfingerprints were further analyzed by DNA sequencing. Twenty-fivedifferent sequences were found, which could be clustered into 16 groupsof antibodies with closely related complementarity determining regions(FIG. 9).

Characterization of scFv Antibodies by Flow Cytometry

The binding specificities of several scFv antibodies were analyzed by3-color flow cytometry (FIG. 7). PBMC isolated from normal donors werestained with FITC-conjugated anti-CD5 and PerCP-conjugated anti-CD19.Staining with scFv antibody was done using biotin-conjugated anti-HA assecondary antibody and PE-conjugated streptavidin. Three antibodies,scFv-2, scFv-3, and scFv-6, were found to specifically recognize theCD19⁺ B lymphocyte population (data not shown). The fourth antibody,scFv-9, recognized two distinct cell populations: the CD19⁺ Blymphocytes and a subset of CD5⁺ T lymphocytes (FIG. 7). Furthercharacterization of the T cell subset showed that it was a subpopulationof the CD4⁺CD8⁻ TH cells (data not shown).

To determine if the antigens recognized by the scFv antibodies wereoverexpressed on primary CLL cells, PBMC from five CLL patients and fivenormal donors were stained with scFv and compared by flow cytometry(FIG. 8 and Table 2). By comparing the mean fluorescent intensities ofthe positive cell populations, the relative expression level of anantigen on CLL cells vs. normal cells could be determined. One antibody,scFv-2, consistently stained CLL cells less intensely than normal PBMC,whereas scFv-3 and scFv-6 both consistently stained CLL cells morebrightly than normal PBMC. The fourth antibody, scFv-9, stained two ofthe five CLL samples much more intensely than normal PBMC, but gave onlymoderately brighter staining for the other three CLL samples (FIG. 8 andTable 2). This indicates that the antigens for scFv-3 and scFv-6 areoverexpressed approximately 2-fold on most if not all CLL tumors,whereas scFv-9 is overexpressed 3 to 6-fold on a subset of CLL tumors.

CLL patients can be divided into two roughly equal groups: those with apoor prognosis (median survival time of 8 years) and those with afavorable prognosis (median survival time of 26 years). Severalunfavorable prognostic indicators have been identified for CLL, mostnotably the presence of VH genes lacking somatic mutations and thepresence of a high percentage of CD38⁺ B cells. Since scFv-9 recognizesan antigen overexpressed in only a subset of CLL patients, we sought todetermine if scFv-9 antigen overexpression correlated with thepercentage of CD38⁺ cells in blood samples from ten CLL patients (FIG.11). The results indicate that scFv-9 antigen overexpression and percentCD38⁺ cells are completely independent of one another.

Identification of Antigens Recognized by scFv Antibodies byImmunoprecipitation (IP) and Mass Spectrometry (MS)

To identify the antigens for these antibodies, scFvs were used toimmunoprecipitate the antigens from lysates prepared from the microsomalfraction of cell-surface biotinylated CLL-AAT cells (FIG. 12). Theimmunoprecipitated antigens were purified by SDS-PAGE and identified bymatrix assisted laser desorption ionization mass spectrometry (MALDI-MS)or microcapillary reverse-phase HPLC nano-electrospray tandem massspectrometry (μLC/MS/MS) (data not shown). ScFv-2 immunoprecipitated a110 kd antigen from both RL and CLL-AAT cells (FIG. 12). This antigenwas identified by MALDI-MS as the B cell-specific marker CD19. ScFv-3and scFv-6 both immunoprecipitated a 45 kd antigen from CLL-AAT cells(not shown). This antigen was identified by MALDI-MS as CD23, which is aknown marker for CLL and activated B cells. ScFv-9 immunoprecipitated a50 kd antigen from CLL-AAT cells (FIG. 12). This antigen was identifiedby μLC/MS/MS as OX-2/CD200, a known marker for B cells, activated CD4⁺ Tcells, and thymocytes. OX-2/CD200 is also expressed on some non-lymphoidcells such as neurons and endothelial cells.

REFERENCES

The following references are incorporated herein by reference to morefully describe the state of the art to which the present inventionpertains. Any inconsistency between these publications below or thoseincorporated by reference above and the present disclosure shall beresolved in favor of the present disclosure.

-   Almasri, N M et al. (1992). Am J Hematol 40 259-263.-   10 Hainsworth, J D (2000). Oncologist 2000; 5(5):376-84-   Nilsson, K (1992). Bum Cell. 5(1):25-41.-   Pu, Q Q and Bezwoda, W (2000). Anticancer Res. 20(4):2569-78.-   Walls A V et al. (1989). Int. J Cancer 44846-853.

It will be understood that various modifications may be made to theembodiments disclosed herein. For example, as those skilled in the artwill appreciate, the specific sequences described herein can be alteredslightly without necessarily adversely affecting the functionality ofthe antibody or antibody fragment. For instance, substitutions of singleor multiple amino acids in the antibody sequence can frequently be madewithout destroying the functionality of the antibody or fragment. Thus,it should be understood that antibodies having a degree of homologygreater than 70% to the specific antibodies described herein are withinthe scope of this disclosure. In particularly useful embodiments,antibodies having a homology greater than about 80% to the specificantibodies described herein are contemplated. In other usefulembodiments, antibodies having a homology greater than about 90% to thespecific antibodies described herein are contemplated. Therefore, theabove description should not be construed as limiting, but merely asexemplifications of preferred embodiments. Those skilled in the art willenvision other modifications within the scope and spirit of thisdisclosure.

1. A method of treating chronic lymphocytic leukemia (CLL) comprisingadministering to a patient suffering from CLL an antibody orantigen-binding fragment thereof that specifically binds to OX-2/CD200.2. The method of claim 1, wherein OX-2/CD200 is overexpressed by CLLcells.
 3. The method of claim 1, wherein said CLL is B-cell chroniclymphocytic leukemia (B-CLL).
 4. The method of claim 1, wherein theantibody or antigen-binding fragment thereof is selected from the groupconsisting of a monoclonal antibody, a human antibody, a humanizedantibody, a chimeric antibody, Fv, scFv, Fab′ and F(ab′)₂.
 5. The methodof claim 4, wherein said humanized antibody or antigen-binding fragmentthereof comprises a framework modification.
 6. The cell line CLL-AATdeposited under ATCC Accession No. PTA-3920.